Project Summary Cell migration drives such key biological events as tissue morphogenesis, immune response, and cancer metastases. Our recent data surprisingly show that the formation and function of the cell leading edge during migration critically depends on arginylation, a relatively unexplored posttranslational modification. Moreover, in cell-based phenotype rescue experiments, we have shown that experimentally arginylated beta actin can largely restore cell leading edge function in mouse embryonic fibroblasts lacking the arginylation enzyme ATE1. In our ongoing studies to determine how N-terminal arginylation of beta actin contributes to lamellipodia formation and directed cell migration, we have made the novel observations that a prominent subset of arginylated beta actin is targeted to the cell leading edge during migration. Moreover, our data demonstrate that beta actin arginylation is selectively regulated by its mRNA sequence rather than its protein structure. In support, gamma actin, which is 99% identical to beta actin at the amino acid level, differs by 13% in its mRNA sequence. We have shown that this difference is directly responsible for faster translation rate of beta actin and leads to its selective arginylation through a novel mechanism coupled to protein ubiquitination. It is also known that zipcode-mediated beta actin mRNA targeting regulates its leading edge localization and, like arginylation, is essential for directional cell migration. We hypothesize that mRNA-mediated regulation of N-terminal arginylation of beta actin uniquely regulates actin function during cell migration by facilitating actin polymerization at the cell leading edge. In this proposal, we will test this hypothesis through three specific aims that will: (1) test the hypothesis that beta actin arginylation facilitates actin polymerization at the cell leading egde; (2) test the hypothesis that beta actin function is uniquely regulated by coding and noncoding regions of its mRNA; and (3) test the hypothesis that this regulation is coupled to modulation of intracellular arginylation activity during cell migration. These experiments will address a novel regulatory mechanism controlling cell polarization and motility through modulating actin's properties and mRNA structure and will ultimately enable a new level of targeted functional studies of cell migration during essential physiological events.